The binding of [3H]inositol 1,4,5-trisphosphate ([3H]IP3) to bovine iris sphincter microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites for [3H]IP3 (Kd=3.52 nM, Bmax=218 fmol/mg protein). The concentration of IP3 receptors in the iris sphincter is much higher than that of the corneal epithelium (99 fmol/mg protein), but considerably lower than that of the rat brain cortex (2250 fmol/mg protein). Kinetic studies on bovine iris sphincter microsomes showed: (a) an extremely rapid time-course of [3H]IP3 binding with a half-maximal binding achieved in 55 sec and reached equilibrium by 10 min; (b) that the [3H]IP3 binding was readily reversible with a t1/2 value of 36 sec, and (c) that the specificity of IP3 receptor sites can be demonstrated by their lack of affinity for 1,3,4-IP3, IP6 and cyclic IP1, and a much weaker affinity for IP1, IP2 and IP4. The results presented provide the first data on the affinity of [3H]IP3 to smooth muscle microsomal membranes. These data support the hypothesis that the [3H]IP3 binding reported here represents a putative physiologically important IP3 receptor which can be quantified in iris sphincter membranes.